1.1 Field of the Invention
The present invention relates to the fields of protein chemistry and immunology; in particular to the isolation and characterization of new calcium binding proteolipids, the encoding DNA and to methods of using the novel proteins for detection of calcifying bacteria in various pathological conditions such as dental calculus and heart valve calcification.
1.2 Description of Related Art
Numerous studies have implicated oral bacteria in the etiology of transient bacteremia and endocarditis (Everett and Hirschmann, 1977). In particular, some studies have indicated that C. matruchotii may play a role in the occurrence of bacterial endocarditis and in the calcification of bicuspid heart valves (Cohen et al., 1992; Iakovidis et al., 1992).
Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. As early as 1925, C. matruchotii (previously known as Leptothrix buccalis and Bacterionema matruchotii) was shown to be present in calcified deposits scraped from teeth (Bulleid, 1925). Subsequently it was demonstrated that these calcium phosphate containing deposits were due to bacteria in the dental calculus and that their production was regulated by various environmental factors (Ennever, 1960; Wasserman et al., 1958; Zander et al., 1960). At the light and electron microscopic level, mineralization in these bacteria has been found to occur either intracellularly, as in Actinomyces israeli, Escherichia coli, Streptococcus sanguis, Streptococcus mutans, Streptococcus salivarius, and some strains of C. matruchotii, or extracellularly, as in Veillonella and the diphtheroids (Ennever etal., 1974; Lie and Selvig, 1974; Rizzo et al., 1962; Streckfuss et al., 1974; Wasserman et al., 1958). The mineralized deposits produce electron diffraction patterns similar to that found in mammalian bone (Boyan-Salyers et al., 1978b; Ennever et al., 1971; Gonzales and Sognnaes, 1960). Also similarly to bone formation (Anderson, 1969), initial deposition of hydroxyapatite in calcifying bacteria has been associated with membranes (Ennever et al., 1968; Ennever et al., 1971; Vogel and Smith, 1976) or membrane components (Boyan and Boskey, 1984; Boyan-Salyers et al., 1978b; Boyan-Salyers and Boskey, 1980; Ennever et al., 1972; Ennever et al., 1976; Ennever et al., 1979).
Calcification of C. matruchotii has been examined using a number of in vitro models (Boyan-Salyers et al., 1978b; Ennever et al., 1971; Lie and Selvig, 1974; Vogel and Smith, 1976). Because mineralization will not occur without an adequate calcium supply, C. matruchotii can be studied under either calcification-permissive or calcification-nonpermissive conditions (Boyan et al., 1984; Boyan-Salyers and Boskey, 1980; Ennever et al., 1971), making it an excellent model for studying mineralization in general, and microbial calcification in particular. The initial steps in apatite formation involve Ca.sup.2+ -binding to acidic phospholipids, particularly phosphoinositides and phosphatidylserine (Boyan-Salyers and Boskey, 1980; Vogel et al., 1978), followed by the addition of inorganic phosphate and Ca.sup.2+ to form apatite [Ca.sub.10 (PO.sub.4).sub.6 ] clusters that are converted by hydration to hydroxyapatite (Vogel and Boyan-Salyers, 1976). It is believed that the acidic phospholipids in the membrane associate with specific proteolipids to form a complex which directs the initial phases of the process (Boyan et al., 1992; Boyan and Boskey, 1984; Boyan-Salyers et al., 1978a; Ennever et al., 1976; Raggio et al., 1986; Vogel and Boyan-Salyers, 1976).
Previous studies have demonstrated that calcifiable proteolipids isolated from C. matruchotii are involved in ion translocation across lipid bilayers. Using reconstituted bacteriorhodopsin-proteoliposomes, translocation of ions across the membrane was greatly enhanced in the presence of proteolipids extracted from C. matruchotii (Boyan et al., 1992; Swain et al., 1989; Swain and Boyan, 1988). Ion-transport across the liposomal membrane was inhibited by dicyclohexylcarbodiimide (DCCD, an inhibitor of proton channels). It has been suggested that proteolipids form an ionophore that could play a role in the intracellular accumulation of calcium and phosphate ions or export of protons, followed by initial mineralization on the inner leaflet of the membrane (Boyan et al., 1989a; 1989b; 1992; Swain and Boyan, 1988; 1989).
A number of studies have shown that calcifiable bacteria contain constituents which can support calcification under appropriate conditions. Membranes isolated from C. matruchotii provide nucleating foci for apatite formation in vitro (Ennever et al., 1976; Vogel and Smith, 1976). More recent data indicate that specific calcifiable proteolipids permit the ordered structuring of phospholipids in the cell membrane so that calcium-acidic phospholipid-phosphate complexes (CPLX) can form (Boyan et al., 1992; Boyan and Boskey, 1984; Boyan-Salyers and Boskey, 1980; Raggio et al., 1986).
Previous work has reported a 8-10 kDa proteolipid involved in C. matruchotii calcification (Boyan, 1985). In an earlier paper, phospholipids were reported to be associated with the protein moiety through hydrophobic interactions (Ennever et al., 1973); with partial removal of this phospholipid resulting in loss of calcifiability (Ennever et al., 1978a; 1978b). Later studies demonstrated the presence of additional proteolipids in the bacteria (Swain et al., 1989), which enhanced ion transport across liposomal membranes.
It has been suggested that proteolipids might function in two capacities during calcification: as sites for CPLX formation and in transport of Ca.sup.2+ and P.sub.i to the calcification site or in the transport of protons away from the site (Boyan et al., 1989a; Swain and Boyan, 1989).
Proteolipids have been reported to play a role in both calcium binding in growth plate cartilage matrix vesicles (Cao et al., 1993; Genge et al., 1991; 1992) and phosphate binding and transport over kidney brush border membranes (Debiec and Lorenc, 1988; Kessler et al., 1982; 1988). In matrix vesicles a nucleational core complex, reminiscent of CPLX, has been reported, consisting of a membrane associated complex of Ca.sup.2+, P.sub.i, phosphatidylserine and annexins, proteins exhibiting proteolipid-like characteristics, and capable of initiating nucleation (Genge et al., 1991; Wu et al., 1993). On the other hand, phosphate transport across kidney brush border membranes has been associated with phosphorin, a 3 kDa membrane proteolipid (Kessler et al., 1982), as well as with a proteolipid-like Na.sup.+,P.sub.i -binding protein with a molecular mass of 155 kDa (Debiec and Lorenc, 1988).
2.0 SUMMARY OF THE INVENTION
The present invention relates to the isolation and characterization of a novel proteolipid "bacteriocalcifin",from C. matruchotii that is involved in the formation of dental calculus ("plaque") and heart valve calcification. The new proteolipid represents a new class of calcium binding species designated "bacteriocalcifins".
The present invention provides biologically active proteolipids comprising the amino acid sequences of bacteriocalcifin-1(SEQ ID NO:1) (5.5 kilodalton proteolipid, designated "bacteriocalcifin-1") and bacteriocalcifin-2 (SEQ ID NO:2) (7.5 kilodalton proteolipid), as well as the nucleotide sequence of the bcf-1 gene for the 5.5 kilodalton proteolipid of (SEQ ID NO:3) and (SEQ ID NO:6) that includes the sequence of (SEQ ID NO:3) encoding a 5.5 kDa bacteriocalcifin. Among the biological properties of the bacteriocalcifins in the present invention is the capability to induce the formation of hydroxyapatite in vivo and the binding of calcium in an in vitro assay system.
An important aspect of the invention concerns the isolation, characterization, amino acid sequencing, cloning and nucleotide sequencing of the proteolipid from C. matruchotii, as well as the assay to determine in vitro calcification activity. The invention also contains the generation of polyclonal and monoclonal antibodies against the proteolipid fraction from C. matruchotii and their use in the detection of the proteolipid in immunoblots, ELISA-assays and in the use of blocking calcium binding activity in an in vitro calcification assay.
The present invention includes the isolation and characterization of a novel protein with an apparent MW of 5.5 kilodaltons after SDS-polyacrylamide gel electrophoresis from the oral bacterium Corynebacterium matruchotii which is involved in the formation of dental calculus ("plaque") and heart valve calcification. Characteristics of the calcium binding protein complex include:
(a) a unique amino acid sequence; PA1 (b) lipid molecules covalently attached to a protein core; PA1 (c) a novel 5.5 kilodalton protein that is a bacterial homolog of a mammalian phosphoprotein phosphatase; and PA1 (d) is involved in calcification of C. matruchotii.
Additionally, the invention describes the generation of polyclonal and monoclonal antibodies against bacterial phosphoprotein phosphatase. The antibodies may be used to detect the presence of C. matruchotii in the oral cavity and in pathological septic calcified deposits. The antibodies are also useful in detecting the presence of phosphoprotein phosphatase and related proteins in cultures of C. matruchotii and other calcifiable bacteria. The antibodies may be used to block the activity of the disclosed calcium binding proteins and are expected to find use in blocking the formation of dental calculus and heart valve calcification.
2.1 Novel Calcium Binding Polypeptides
In an important aspect therefore, the present invention relates to the discovery of a novel proteolipid calcium-binding protein isolated from Cornyebacterium matruchotii. The intact proteolipid comprises three apoproteins covalently attached to a lipid and has an apparent molecular weight of approximately 10 kDa. The proteolipid polypeptide components do not show substantial homology with calcifying strains of Streptococcus, e.g., S. sanguis, type II.
One of the three apoproteins has an apparent molecular weight of 5.5 kDa by SDS PAGE and appears to be a bacterial homolog of mammalian pliosphoprotein phosphatase. The amino acid sequence of the apoprotein has been determined in accordance with SEQ ID NO:1 and an N-terminal sequence in accordance with SEQ ID NO:5.
A second apoprotein has an apparent molcular weight of 7.5 kDa by SDS-PAGE. A partial peptide sequence (SEQ ID NO:2) represents the N-terminal sequence.
The third apoprotein has an apparent molecular weight of 5.0 kDa by SDS-PAGE. Its amino acid sequence is in accordance with SEQ ID NO:8. The partial amino acid sequence representing the N-terminal sequence is represented by SEQ ID NO:5.
2.2 Pharmaceutical Compositions
Another aspect of the present invention includes novel compositions comprising isolated and purified apoproteins, proteolipids or nucleic acids which encode the disclosed proteolipid calcium binding protein. Regarding nucleic acids, it will, of course, be understood that one or more than one calcium-binding proteolipid gene may be used in the methods and compositions of the invention. The nucleic acid delivery methods may thus entail the administration of one, two, three, or more, homologous genes. The maximum number of genes that may be applied is limited only by practical considerations, such as the effort involved in simultaneously preparing a large number of gene constructs or even the possibility of eliciting an adverse cytotoxic effect.
With regard to the calcium-binding protein and proteolipid compositions, it is contemplated that such compositions will contain a biologically effective amount of the novel peptide, peptides or lipid associated forms of such peptides. As used herein a "biologically effective amount" of a peptide or composition refers to an amount effective to stimulate or promote calcium binding. As disclosed herein, different peptide amounts may be effective, as shown in vitro, such as may be effective in vivo between about 6 to about 11 mg/kg.
Clinical doses will of course be determined by the nutritional status, age, weight and health of the patient. The quantity and volume of the peptide composition administered will depend on the subject and the route of administration. The precise amounts of active peptide required will depend on the judgment of the practitioner and may be peculiar to each individual. However, in light of the data presented herein, the determination of a suitable dosage range for use in humans will be straightforward.
The compositions for use in stimulating antibodies for blocking calcium binding in accordance with the present invention will be compositions that contain the full length peptide or partial sequences including effective epitopes. The term "a peptide" or "a polypeptide" in this sense means at least one peptide or polypeptide which includes a sequence of any of the aforementioned structures or variants thereof. The terms peptide, polypeptide, or protein may be used interchangeably.
In addition to including an amino acid sequence in accordance with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:8, the peptides may include various other shorter or longer fragments or other short peptidyl sequences of various amino acids. In certain embodiments, the peptides may include shorter sequences, for example the N-terminal regions, SEQ ID NO:5 or SEQ ID:6, of the apoprotein or additional sequences such as short targeting sequences, tags, labelled residues, amino acids contemplated to increase the half life or stability of the peptide or any additional residue for a designated purpose, so long as the peptide still functions as a calcium binding agent and as such will stimulate antibodies to block this activity. Such functionality may be readily determined by assays such as those described herein.
Any of the commonly occurring amino acids may be incorporated into the peptides, including alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Likewise, any of the so-called rare or modified amino acids may also be incorporated into a peptide of the invention, including: 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine (beta-Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid), 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 2,2'-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isoeesmosine, allo-Isoleucine, N-Methylglycine sarcosine), N-Methylisoleucine, N-Methylvaline, Norvaline, Norleucine and Ornithine.
The inhibitory compositions of the invention may include a peptide modified to render it biologically protected. Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Pat. No. 5,028,592, incorporated herein by reference, protected peptides often exhibit increased pharmacological activity.
Compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof. The use of D-amino acids may confer additional resistance to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
Likewise, compositions that make use of calcium-binding proteolipid encoding genes are also contemplated. The particular combination of genes may be two or more variants of such genes; or it may be such that a calcium binding proteolipid gene is combined with another gene and/or another protein such as a alkaline, neutral or acid phosphatase, cofactor or other biomolecule; a hormone or growth factor gene may even be combined with a gene encoding a cell surface receptor capable of interacting with the polypeptide product of the first gene.
In using multiple genes, they may be combined on a single genetic construct under control of one or more promoters, or they may be prepared as separate constructs of the same or different types. Thus, an almost endless combination of different genes and ogenetic constructs may be employed. Certain gene combinations may be designed to, or their use may otherwise result in, achieving synergistic effects on cell growth and/or stimulation of an immune response. Any and all such combinations are intended to fall within the scope of the present invention. Indeed, many synergistic effects have been described in the scientific literature, so that one of ordinary skill in the art would readily be able to identify likely synergistic gene combinations, or even gene-protein combinations.
It will also be understood that, if desired, the nucleic acid segment or gene encoding a calcium binding proteolipid could be administered in combination with further agents, such as, e.g., proteins or polypeptides or various pharmaceutically active agents. So long as the composition comprises a calcium-binciing proteolipid gene, there is virtually no limit to other components which may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The nucleic acids may thus be delivered along with various other agents as required in the particular instance.
Pharmaceutical compositions prepared in accordance with the present invention find use in several applications, including blocking of the formation of dental calculus and prevention of heart valve calcification. Such methods generally involve administering to a mammal a pharmaceutical composition comprising an immunologically effective amount of a calcium-binding proteolipid or apoprotein composition. This composition may include an immunologically-effective amount of either the apo- or lipoproteins herein described or their corresponding encoding nucleic acid composition. Such compositions would typically stimulate an immune response in a mammal.
Therapeutic kits comprising the aforementioned proteolipids, apoprotein components or corresponding-encoding nucleic acid segments comprise another aspect of the present invention. Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of a calcium-binding proteolipid, apoprotein or the encoding nucleic acid composition. The kit may have a single container means that contains the polypeptide composition or it may have distinct container means for the compositions and other reagents which may be included within such kits.
The components of the kit may be provided as liquid solution(s), or as dried powder(s). When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. When reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
In related embodiments, the present invention contemplates the preparation of diagnostic kits that may be employed to detect the presence of calcium-binding proteins or peptides and/or antibodies in a sample. Generally speaking, kits in accordance with the present invention will include a suitable calcium-binding protein or peptide or antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The components of the diagnostic kits may be packaged either in aqueous media or in lyophilized form.
The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention. The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
The container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen or antibody may be placed, and preferably suitably aliquoted. Where a second binding ligand is provided, the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
2.3 Antibodies
In another aspect, the present invention includes an antibody that is immunoreactive with a polypeptide of the invention. An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Howell and Lane, 1988).
Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
Antibodies, both polyclonal and monoclonal, specific for the proteolipids or apoproteins associated with the proteolipids may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. A composition containing antigenic epitopes of the proteins can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the protein. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
To obtain monoclonal antibodies, one would also initially immunize an experimental animal, often preferably a mouse, with a calcium-binding proteolipid or apoprotein of such a proteolipid composition. One would then, after a period of time sufficient to allow antibody generation, obtain a population of spleen or lymph cells from the animal. The spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.
Following immunization, spleen cells are removed and fused, using a standard fusion protocol with plasmacytoma cells to produce hybridomas secreting monoclonal antibodies against the calcium-binding proteolipid. Hybridomas which produce monoclonal antibodies to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods. Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the calcium-binding proteolipid-specific monoclonal antibodies.
It is proposed that the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures which may utilize antibody specific to calcium-binding proteolipid epitopes.
Additionally, it is proposed that monoclonal antibodies specific to the particular chemokine may be utilized in other useful applications. For example, their use in immunoabsorbent protocols may be useful in purifying native or recombinant calcium-binding proteolipids from other bacterial species or variants thereof.
In general, both poly- and monoclonal antibodies against the proteolipids herein disclosed may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding the proteolipid or related proteins. They may also be used in inhibition studies to analyze the effects of the proteolipid in cells or animals. Anti-calcium-binding proteolipid antibodies will also be useful in immunolocalization studies to analyze the distribution of calcium binding proteolipids during various cellular events, for example, to determine the cellular or tissue-specific distribution of such peptides under different physiological conditions. A particularly useful application of antibodies generated from the proteolipids is in purifying native or recombinant calcium-binding proteolipids, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
2.7 Recombinant Polypeptides
Recombinant versions of a protein or polypeptide are deemed as part of the present invention. Thus one may, using techniques familiar to those skilled in the art, express a recombinant version of the polypeptide in a recombinant cell to obtain the polypeptide from such cells. The techniques are based on cloning of a DNA molecule encoding the polypeptide from a DNA library, that is, on obtaining a specific DNA molecule distinct from other DNAs. One may, for example, clone a cDNA molecule, or clone genomic DNA. Techniques such as these would also be appropriate for the production of the bacteriocalcifin polypeptides in accordance with the present invention.
2.8 Genes
As known to those of skill in the art, the original source of a recombinant gene or DNA segment to be used in a therapeutic regimen need not be of the same species as the animal to be treated. In this regard, it is contemplated that any recombinant calcium-binding proteolipid gene may be employed in the methods disclosed herein such as the identification of cells containing DNA encoding calcium-binding proteolipid or variants of the protein.
Particularly preferred genes are those isolated from bacteria, particularly C. matruchotii as well as closely related species, including other oral bacteria, such as Actinomyces israeli, Streptococcus sanguis, S. mitis, S. salivarius, Veillonella, the diptheroids, and certain strains of Escherichia coli. It is contemplated that homologous genes encoding proteolipids of similar calcium binding activity will be found in such other related species as C. glutamicum. Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium pseudotuberculosis. However, since the sequence homology for genes encoding the protein may be conserved across species lines, equine, murine, and bovine species may also be contemplated as sources, in that such genes and DNA segments are readily available; however, with the bacterial forms of the gene being most preferred for use in treatment regimens. Recombinant proteins and polypeptides encoded by isolated DNA segments and genes are often referred to with the prefix "r" for recombinant and "rh" for recombinant human. As such, DNA segments encoding rcalcium-binding proteolipids, or rcalcium-binding proteolipid-related genes, etc. are contemplated to be particularly useful in connection with this invention. Any recombinant proteolipid gene would likewise be very useful with the methods of the invention.
The definition of a "calcium-binding proteolipid gene", as used herein, is a gene that hybridizes, under relatively stringent hybridization conditions (see, e.g., Maniatis et al., 1982), to DNA sequences presently known to include calcium-binding proteolipid gene sequences.
To prepare a calcium-binding proteolipid gene segment or cDNA one may follow the teachings disclosed herein and also the teachings of any of patents or scientific documents specifically referenced herein. One may obtain a rcalcium-binding proteolipid-encoding DNA segments using molecular biological techniques, such as polymerase chain reaction (PCR) or screening of a cDNA or genomic library, using primers or probes with sequences based on the above nucleotide sequence. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Pat. Nos. 4,683,195 and 4,683,202 (herein incorporated by reference). The practice of these techniques is a routine matter for those of skill in the art, as taught in various scientific texts (see e.g., Sambrook et al., 1989), incorporated herein by reference. Certain documents further particularly describe suitable mammalian expression vectors, e.g., U.S. Pat. No. 5,168,050, incorporated herein by reference. The genes and DNA segments that are particularly preferred for use in certain aspects of the present methods are those encoding bacterial calcium-binding proteolipids and related polypeptides.
It is also contemplated that one may clone further genes or cDNAs that encode a calcium-binding peptide, protein or polypeptide. The techniques for cloning DNA molecules, i.e., obtaining a specific coding sequence from a DNA library that is distinct from other portions of DNA, are well known in the art. This can be achieved by, for example, screening an appropriate DNA library which relates to the cloning of a calcium-binding gene such as the C. matruchotii proteolipids disclosed herein. The screening procedure may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of known DNA sequences encoding related cytokine proteins. The operation of such screening protocols are well known to those of skill in the art and are described in detail in the scientific literature, for example, see Sambrook et al., 1989.
Techniques for introducing changes in nucleotide sequences that are designed to alter the functional properties of the encoded proteins or polypeptides are well known in the art, e.g., U.S. Pat. No. 4,518,584, incorporated herein by reference, which techniques are also described in further detail herein. Such modifications include the deletion, insertion or substitution of bases, and thus, changes in the amino acid sequence. Changes may be made to increase the cytokine activity of a protein, to increase its biological stability or half-life, to change its glycosylation pattern, and the like. All such modifications to the nucleotide sequences are encompassed by this invention.
2.8.1 Calcium-binding Proteolipid-Encoding DNA Segments
The present invention, in a general and overall sense, also concerns the isolation and characterization of a novel C. matruchotii calcium-binding proteolipid gene which encodes the apoprotein portion of the 10 kDa proteolipid isolated from C. matruchotii . A preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein that has at least a partial amino acid sequence in accordance with SEQ ID NO:1. Another embodiment of the present invention is a purified nucleic acid segment, further defined as including a nucleotide sequence in accordance with SEQ ID NO:3.
In a more preferred embodiment the purified nucleic acid segment consists essentially of the nucleotide sequence of SEQ ID NO:3 its complement and the degenerate variants thereof. As used herein, the term "nucleic acid segment" and "DNA segment" are used interchangeably and refer to a DNA molecule which has been isolated free of total genomic DNA of a particular species. Therefore, a "purified" DNA or nucleic acid segment as used herein, refers to a DNA segment which contains a calcium-binding proteolipid coding sequence yet is isolated away from, or purified free from, total genomic DNA, for example, total cDNA or human genomic DNA. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
Similarly, a DNA segment comprising an isolated or purified bcf gene refers to a DNA segment including calcium-binding proteolipid coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences. In this respect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences or combinations thereof. "Isolated substantially away from other coding sequences" means that the gene of interest forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a calcium-binding proteolipid gene, that includes within its amino acid sequence an amino acid sequence in accordance with SEQ ID NO:1. In other embodiments, the amino acid sequence included may be that of SEQ ID NO:2, SEQ ID NO:5, or SEQ ID NO:8. Moreover, in other particular embodiments, the invention concerns isolated DNA segments and recombinant vectors incorporating DNA sequences which encode a gene that includes within its amino acid sequence the amino acid sequence of a calcium-binding proteolipid gene corresponding to homologous genes in other species, particularly bacterial species related to C. matruchotii.
Another preferred embodiment of the present invention is a purified nucleic acid segment that encodes a protein in accordance with SEQ ID NO:3, further defined as a recombinant vector. As used herein the term, "recombinant vector", refers to a vector that has been modified to contain a nucleic acid segment that encodes a calcium-binding proteolipid, or a fragment thereof. The recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said calcium-binding proteolipid-encoding nucleic acid segment.
A further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising a calcium binding protein encoding gene. The recombinant host cell may be a prokaryotic cell. In a more preferred embodiment, the recombinant host cell is a eukaryotic cell. As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding calcium-binding proteolipids, has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. Howvever, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
In certain embodiments, the invention concerns isolated DNA segments and recombinant vectors which encode a protein or peptide that includes within its amino acid sequence an amino acid sequence essentially as set forth in SEQ ID NO:1 or in SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:8. Naturally, where the DNA segment or vector encodes a full length bacteriocalcifin protein, or is intended for use in expressing the protein, the most preferred sequences are those which are essentially as set forth in SEQ ID NO:1 or SEQ ID NO:8. It is recognized that SEQ ID Nos:2,5 and 6 represent partial amino acid sequences at the N-terminus of the apoproteins comprising the protein-lipid complex isolated from C. matruchotii but which however are encoded by the isolated gene and as such are contemplated embodiments which also include up to the full length sequence of each apoprotein and functional variants as well.
The term "a sequence essentially as set forth in SEQ ID NO:1" or in reference to any other sequence referred to herein, means that the sequence substantially corresponds to a portion of SEQ ID NO:1 and has relatively few amino acids which are not identical to, or a biologically functional equivalent of, the amino acids of SEQ ID NO:1. The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEQ ID NO:3, and that is associated with a calcium-binding proteolipid. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81% and about 90%; or even more preferably, between about 91%, 95% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of SEQ ID NO:1 will be sequences which are "essentially as set forth in SEQ ID NO:1"
In certain other embodiments, the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ ID NO:3. The term "essentially as set forth in SEQ ID NO:3," is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:3, and has relatively few codons which are not identical, or functionally equivalent, to the codons of SEQ ID NO:3. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, as set forth in Table 1, and also refers to codons that encode biologically equivalent amino acids.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
Excepting intronic or flanking regions, and allowing for the degeneracy of the genetic code, sequences which have between about 70% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:3 will be sequences which are "essentially as set forth in SEQ ID NO:3". Sequences which arc essentially the same as those set forth in SEQ ID NO:3 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:3 under relatively stringent conditions. Suitable relatively stringent hybridization conditions will be well known to those of skill in the art and are clearly set forth herein, for example conditions for use with Southern and Northern blot analysis, and as described in examples herein set forth.
Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:3. Nucleic acid sequences which are "complementary" are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to the nucleic acid segment of SEQ ID NO:3 under relatively stringent conditions that may also be understood as including conditions of high stringency.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared which include a short stretch complementary to SEQ ID NO:3, such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which are up to 200 or so base pairs in length. DNA segments with total lengths of about 500, 200, 100 and about 50 base pairs in length are also contemplated to be useful.
A preferred embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:3. In a more preferred embodiment the nucleic acid is further defined as comprising at least a 20 nucleotide long stretch, a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotide long stretch, or at least a 200 nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:3. The nucleic acid segment may be further defined as having the nucleic acid sequence of SEQ ID NO:3.
A related embodiment of the present invention is a nucleic acid segment which comprises at least a 14-nucleotide long stretch which corresponds to, or is complementary to, the nucleic acid sequence of SEQ ID NO:3, further defined as comprising a nucleic acid fragment of up to 10,000 basepairs in length. A more preferred embodiment if a nucleic acid fragment comprising from 14 nucleotides of SEQ ID NO:3 up to 5,000 basepairs in length, 3,000 basepairs in length, 1,000 basepairs in length, 500 basepairs in length, or 100 basepairs in length.
Naturally, it will also be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of SEQ ID NO:3. Recombinant vectors and isolated DNA segments may therefore variously include the calcium-binding proteolipid coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides which nevertheless include calcium-binding proteolipid-coding regions or may encode biologically functional equivalent proteins or peptides which have variant amino acids sequences.
The DNA segments of the present invention encompass biologically functional equivalent calcium-binding proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the calcium-binding proteolipids or to test mutants in order to examine activity or determine the presence of calcium-binding proteolipids in various cells and tissues at the molecular level.
A preferred embodiment of the present invention is a purified composition comprising a polypeptide having an amino acid sequence in accordance with any of SEQ ID Nos:1, 2, 5,6,or 7. The term "purified" as used herein, is intended to refer to a calcium-binding proteolipid composition, wherein the lipoprotein or any of the apoprotein components is purified to any degree relative to its naturally-obtainable state, i.e., in this case, relative to its purity within a eukaryotic cell extract. A preferred cell for the isolation of the proteins is a bacterial cell, such as C. matruchotii and related species; however, the proteolipid might also be isolated from patient specimens, recombinant cells, tissues, isolated subpopulations of tissues, and the like, as will be known to those of skill in the art, in light of the present disclosure. A purified calcium-binding proteolipid composition therefore also refers to a polypeptide having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:8 free from the environment in which it may naturally occur.
If desired, one may also prepare fusion proteins and peptides, e.g., where the calcium-binding proteolipid coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins which may be purified by affinity chromatography and enzyme label coding regions, respectively).
Turning to the expression of the calcium-binding proteolipid gene whether from cDNA based or genomic DNA, one may proceed to prepare an expression system for the recombinant preparation of any one or more of the apo proteins of the calcium-binding proteolipid. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. For example, one may prepare a fusion protein that combines GST (glutathione-S-transferase) with the protein of SEQ ID NO:1 or SEQ ID NO:8 or a sequence including any or all of the apoprotein sequences that may be included in a calcium-binding proteolipid fusion protein. This may be a convenient means of bacterial expression. However, it is believed that virtually any expression system may be employed in the expression of such calcium-binding proteolipids.
Another embodiment is a method of preparing a protein composition comprising growing recombinant host cell comprising a vector that encodes a protein which includes an amino acid sequence in accordance with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 or SEQ ID 7, under conditions permitting nucleic acid expression and protein production followed by recovering the protein so produced. The host cell, conditions permitting nucleic acid expression, protein production and recovery, will be known to those of skill in the art, in light of the present disclosure of the encoding gene.
2.8.2 Gene Constructs and DNA Segments
As used herein, the terms "gene" and "DNA segment" are both used to refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a gene or DNA segment encoding a calcium-binding proteolipid refers to a DNA segment that contains sequences encoding a calcium-binding protelipid, but is isolated away from, or purified free from, total genomic DNA of the species from which the DNA is obtained. Included within the term "DNA segment", are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, retroviruses, adenoviruses, and the like.
The term "gene" is used for simplicity to refer to a functional protein or peptide encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences and cDNA sequences. "Isolated substantially away from other coding sequences" means that the gene of interest, in this case, a calcium-binding proteolipid gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or cDNA coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions, such as sequences encoding leader peptides or targeting sequences, later added to the segment by the hand of man.
2.8.3 Recombinant Vectors Expressing Calcium-binding Proteolipid Protein
A particular aspect of this invention provides novel ways in which to utilize calcium-binding proteolipid-encoding DNA segments and recombinant vectors comprising DNA segments encoding the component proteins of the proteolipid. As is well known to those of skill in the art, many such vectors are readily available. One particular detailed example of a suitable vector for expression in mammalian cells is that described in U.S. Pat. No. 5,168,050, incorporated herein by reference. However, there is no requirement that a highly purified vector be used, so long as the coding segment employed encodes a calcium-binding protein and does not include any coding or regulatory sequences that would have an adverse effect on cells. Therefore, it will also be understood that useful nucleic acid sequences may include additional residues, such as additional non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
After identifying an appropriate calcium-binding proteolipid-encoding gene or DNA molecule, it may be inserted into any one of the many vectors currently known in the art, so that it will direct the expression and production of the calcium-binding protein when incorporated into a host cell. In a recombinant expression vector, the coding portion of the DNA segment is positioned under the control of a promoter. The promoter may be in the form of the promoter which is naturally associated with a calcium-binding protein-encoding gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR technology, in connection with the compositions disclosed herein.
In certain embodiments, it is contemplated that particular advantages will be gained by positioning the bacteriocalcifin-encoding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a calcium-binding proteolipid gene in its natural environment. Such promoters may include those normally associated with other calcium-binding polypeptide genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the particular cell containing the vector comprising the calcium-binding protein gene.
The use of recombinant promoters to achieve protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., (1989). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level or regulated expression of the introduced DNA segment. The currently preferred promoters are those such as CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter in combination with the SV40 enhancer.
2.9 Methods of DNA Transfection
Technology for introduction of DNA into cells is well-known to those of skill in the art. Four general methods for delivering a gene into cells have been described: (1) chemical methods (Graham and Van der Eb, 1973); (2) physical methods such as microinjection (Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and the gene gun (Yang et al., 1990); (3) viral vectors (Clapp, 1993; Danos and Heard, 1992; Eglitis and Anderson, 1988); and (4) receptor-mediated mechanisms (Wu et al., 1991; Curiel et al., 1991; Wagner et al., 1992).
2.9.1 Liposomes and Nanocapsules
The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al., 1991 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half-times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987). The following is a brief description of these DNA delivery modes.
Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al., 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 .mu.m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made, as described (Couvreur et al., 1984; 1988).
Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 .ANG., containing an aqueous solution in the core.
In addition to the teachings of Couvreur et al. (1991), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
2.10 Expression of Calcium-binding Protein
For the expression of calcium-binding protein, once a suitable (full-length if desired) clone or clones have been obtained, whether they be cDNA based or genomic, one may proceed to prepare an expression system for the recombinant preparation of calcium-binding protein. The engineering of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression. It is believed that virtually any expression system may be employed in the expression of calcium-binding protein.
Calcium-binding proteolipids may be successfully expressed in eukaryotic expression systems, however, it is also envisioned that bacterial expression systems may be used for the preparation of bacteriocalcifin proteins for virtually all purposes. The cDNA for bacteriocalcifin protein may be separately expressed in bacterial systems, with the encoded proteins being expressed as fusions with .beta.-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, green fluorescent protein and the like. It is believed that bacterial expression will ultimately have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained thereby.
It is proposed that transformation of host cells with DNA segments encoding bacteriocalcifins will provide a convenient means for obtaining calcium-binding proteins and peptides. Both cDNA and genomic sequences are suitable for eukaryotic expression, as the host cell will, of course, process the genomic transcripts to yield functional mRNA for translation into protein.
It is similarly believed that almost any eukaryotic expression system may be utilized for the expression of calcium-binding protein, e.g., baculovirus-based, glutamine synthase-based or dihydrofolate reductase-based systems could be employed. However, in preferred embodiments, it is contemplated that plasmid vectors incorporating an origin of replication and an efficient eukaryotic promoter, as exemplified by the eukaryotic vectors of the pCMV series, such as pCMV5, will be of most use.
For expression in this manner, one would position the coding sequences adjacent to and under the control of the promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame of the protein between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter.
Where eukaryotic expression is contemplated, one will also typically desire to incorporate into the transcriptional unit which includes calcium-binding protein, an appropriate polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained within the original cloned segment. Typically, the poly A addition site is placed about 30 to 2000 nucleotides "downstream" of the termination site of the protein at a position prior to transcription termination.
Translational enhancers may also be incorporated as part of the vector DNA. Thus the DNA constructs of the present invention should also preferable contain one or more 5' non-translated leader sequences which may serve to enhance expression of the gene products from the resulting mRNA transcripts. Such sequences may be derived from the promoter selected to express the gene or can be specifically modified to increase translation of the RNA. Such regions may also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence (Griffiths, et al, 1993).
Such "enhancer" sequences may be desirable to increase or alter the translational efficiency of the resultant mRNA. The present invention is not limited to constructs where the enhancer is derived from the native 5'-nontranslated promoter sequence, but may also include non-translated leader sequences derived from other non-related promoters such as other enhancer transcriptional activators or genes.
It is contemplated that virtually any of the commonly employed host cells can be used in connection with the expression of calcium-binding protein in accordance herewith. Examples include cell lines typically employed for eukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI 38, BHK, COS-7, RIN and MDCK cell lines.
It is contemplated that calcium-binding protein may be "overexpressed", i.e., expressed in increased levels relative to its natural expression in human cells, or even relative to the expression of other proteins in a recombinant host cell containing calcium-binding protein-encoding DNA segments. Such overexpression may be assessed by a variety of methods, including radio-labeling and/or protein purification. However, simple and direct methods are preferred, for example, those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analyses, such as densitometric scanning of the resultant gel or blot. A specific increase in the level of the recombinant protein or peptide in comparison to the level in natural calcium-binding protein-producing animal cells is indicative of overexpression, as is a relative abundance of the specific protein in relation to the other proteins produced by the host cell and, e.g., visible on a gel.
As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a calcium-binding peptide has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man. Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
It will be understood that recombinant calcium-binding protein may differ from naturally produced calcium-binding protein in certain ways. In particular, the degree of post-translational modifications, such as, for example, glycosylation and phosphorylation may be different between the recombinant calcium-binding protein and the calcium binding polypeptide purified from a natural source, such as calcifying bacteria.
Generally speaking, it may be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, the inventors do not exclude the possibility of employing a genomic version of a particular gene where desired.
After identifying an appropriate DNA molecule by any or a combination of means as described above, the DNA may then be inserted into any one of the many vectors currently known in the art and transferred to a prokaryotic or eukaryotic host cell where it will direct the expression and production of the so-called "recombinant" version of the protein. The recombinant host cell may be selected from a group consisting of S. mutans, E. coli, S. cerevisae. Bacillus sp., Lactococci sp., Enterococci sp., or Salmonella sp. In certain preferred embodiments, the recombinant host cell will have a recA phenotype.
Where the introduction of a recombinant version of one or more of the foregoing genes is required, it will be important to introduce the gene such that it is under the control of a promoter that effectively directs the expression of the gene in the cell type chosen for engineering. In general, one will desire to employ a promoter that allows constitutive (constant) expression of the gene of interest. Commonly used constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, and the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes. The level of expression from the introduced genes of interest can vary in different clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA. Thus, the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
2.10.1 Enhanced Production of Calcium-binding Protein
One of the problems with calcium-binding proteins isolated from natural sources is low yields and extensive purification processes. An aspect of the present invention is the enhanced production of calcium-binding protein by recombinant methodologies in a bacterial host, employing DNA constructs to transform Gram-positive or Gram-negative bacterial cells. For example, the use of Escherichia coli expression systems is well known to those of skill in the art, as is the use of other bacterial species such as Bacillus subtilis or Streptococcus sanguis.
Further aspects of the invention include high expression vectors incorporating DNA encoding the novel bacteriocalcifin and its variants. It is contemplated that vectors providing enhanced expression of bacteriocalcifin in other systems such as S. mutans will also be obtainable. Where it is desirable, modifications of the physical properties of bacteriocalcifin may be sought to increase its solubility or expression in liquid culture. The bcf locus may be placed under control of a high expression promoter or the components of the expression system altered to enhance expression.
In further embodiments, the DNA encoding the bacteriocalcifin of the present invention allows for the large scale production and isolation of the bacteriocalcifin polypeptide. This can be accomplished by directing the expression of the mutacin polhpeptide by cloning the DNA encoding the bacteriocalcifin polypeptide into a suitable expression vector. Such an expression vector may then be transformed into a host cell that is able to produce the bacteriocalcifin protein. The bacteriocalcifin protein may then be purified, e.g., by means provided for in this disclosure and utilized in a biologically active form. Non-biologically active recombinant bacteriocalcifin may also have utility, e.g., as an immunogen to prepare anti-bacteriocalcifin antibodies.
2.10.3 Cloning of Calcium-binding Protein Gene
In still another embodiment, the present disclosure provides methods for cloning the DNA encoding the calcium-binding polypeptide. Using methods well known to those of skill in the art, the DNA that encodes the purified calcium-binding protein of the present invention may be isolated and purified. For example, by designing a degenerate oligonucleotide comprising nucleotides complementary to the DNA encoding sequence of SEQ ID NO:1 or SEQ ID NO:8, the calcium-binding protein-encoding DNA can be cloned from a C. matruchotii cell library. Such sequences have been designed based on the N-terminal sequences of these sequences, i.e., SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
The DNA sequences disclosed by the invention allow for the preparation of relatively short DNA (or RNA) sequences which have the ability to specifically hybridize to a gene encoding the calcium-binding polypeptides. Such a gene, is here termed the bcf gene and is understood to mean the gene locus encoding the calcium-binding proteolipid protein structural gene. In these aspects, nucleic acid probes of an appropriate length are prepared. Such probes are typically prespred based on the consideration of the defined amino acid sequence of purified calcium-binding protein. The ability of such nucleic acid probes to specifically hybridize to cbp gene sequences lend them particular utility in a variety of embodiments. For example, the probes may be used in a variety of diagnostic assays for detecting the presence of cbp genes in oral mucosal samples; however, other uses are envisioned, including identification of bcf gene sequences encoding similar or mutant polypeptides related to the bacteriocalcifin. Other uses include the use of mutant species primers, or primers to prepare other genetic constructs.
A first step in such cloning procedures is the screening of an appropriate DNA library, such as, in the present case, genomic or cDNA prepared from an appropriate cell library; for example, C. matruchotii cells. The screening procedure may be an expression screening protocol employing antibodies directed against the protein, or activity assays. Alternatively, screening may be based on the hybridization of oligonucleotide probes, designed from a consideration of portions of the amino acid sequence of the protein, or from the DNA sequences of genes encoding related proteins. Another cloning approach contemplated to be particularly suitable is the use of a probe or primer directed to a gene known to be generally associated with, e.g., within the same operon as, the structural gene that one desires to clone.
Another approach toward identifying the gene(s) responsible for the production of calcium-binding protein is to locate genes known to be adjacent to related calcium-binding protein genes. From sequenced loci in genes that encode similarly functional peptides, it will be possible to determine if these genes share areas of common sequences. A series of oligonucleotide primers complementary to conserved sequences could be used in PCR reactions to amplify the intervening sequence, this amplicon could be used as a probe to identify putative bacteriocalcifin genes. PCR technology is described in U.S. Pat. No. 4,603,102, incorporated herein by reference. Where such a bacteriocalcifin gene is found to be part of every known calcium-binding protein gene, the structural gene for calcium-binding protein should be nearby and readily identified by a technique known as "chromosome walking".
Antibodies against the proteolipid from C. matruchotii have immunologically detected the presence of proteolipid in calcified heart valves. Antibodies against the bacterial proteolipids may play a role as therapeutic agents in the detection, treatment and prevention of bacterial endocarditis and calcification of bicuspid heart valves.
A proteolipid from Corynebacterium matruchotii has calcification activity and a molecular weight of less than 10 kilodaltons. The present invention provides biologically active proteolipids comprising the amino acids sequences of bacteriocalcifin-1 (SEQ ID NO:1) and bacteriocalcifin-2 (SEQ ID NO:2), as well as the nucleotide sequence of the gene for the 5.5 kilodalton proteolipid of (SEQ ID NO:3) Among the biological properties of the proteolipids in the present invention is the capability to induce the formation of hydroxyapatite in vivo, in vitro from a metastable calcium phosphate solution, and the binding of calcium in an in vitro assay.
The purification is a process comprising culturing C. matruchotii in medium comprising calcium; extracting proteolipid from the cultured C. matruchotii cells with a chloroform:methanol mixture; precipitating the proteolipid from the chloroform:methanol extract with acetone and/or diethyl ether; and hydrophobic interaction chromatography on SEPHADEX.TM. LH-20 using chloroform:methanol as the mobile phase. This proteolipid comprises a protein with an N-terminal amino acid sequence of Ala-Gly-Val-Pro-Gly-Val (SEQ ID NO:4). The proteolipid has a more extensive N-terminal amino acid sequence of (SEQ ID NO:2).
The proteolipid preparation comprises, after delipidation, delipidated proteolipids (apoproteins) having molecular weights of about 7.5 kilodaltons, about 5.5 kilodaltons, and about 5.0 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 7.5 kilodalton apoprotein component of the C. matruchotii 10 kilodalton proteolipid preparation has an N-terminal amino acid sequence of (SEQ ID NO:2).
The 5.5 kilodalton apoprotein has an N-terminal amino acid sequence of (SEQ ID NO:5). The 5.5 kilodalton apoprotein component of the 10 kilodalton C. matruchotii proteolipid preparation has a sequence of 50 amino acids (SEQ ID NO:1).
The 5.0 kilodalton apoprotein has an N-terminal amino acid sequence of (SEQ ID NO:5). The 5.0 kilodalton apoprotein component of the 10 kilodalton C. matruchotii proteolipid preparation has a sequence of 47 amino acids (SEQ ID NO:8).
The invention also comprises the cDNA nucleotide sequence for the 5.5 kilodalton apoprotein and any other nucleotide gene sequence that contains the nucleotide sequence for the gene of the 5.5 kilodalton apoprotein, as well as any of the oligonucleotide primers, based on the amino acid sequence of the 5.5 kilodalton aproproteolipid, used to generate these sequences by use of PCR.TM. technique. The 5.5 kilodalton apoprotein has a nucleotide sequence of (SEQ ID NO:3).
Also part of the present invention are polyclonal and monoclonal antibodies directed against the 10 kilodalton proteolipid preparation of C. matruchotii, as well as the use of these polyclonal and monoclonal antibodies diagnostically and therapeutically. The antibodies of the present invention are useful in detecting the presence of C. matruchotii and other calcifying microorganism, such as, but not limited to, Escherichia coli strains and Streptococcus sanguis, that produce substantially homologous proteolipids that are involved in calcification and can specifically be detected by the before-mentioned antibodies. The polyclonal and monoclonal antibodies are also useful in inhibiting the formation of dental calculus and calcification of heart valves. A method of the present invention is for inhibiting dental calculus and heart valve calcification and comprises inducing immunity to the 10 kilodalton proteolipid preparation of C. matruchotii.
The present invention also provides compositions, such as diagnostic, therapeutical, and pharmaceutical compositions, containing the proteolipid preparation or any of its apoprotein components of the present invention as well as antibodies against the proteolipid preparation of the present invention and methods of using either the proteolipid and/or the antibodies in treatment and diagnosis.
Other and further objects features and advantages will be apparent from the following description of the presently preferred embodiments of the invention, given for the purposes of disclosure when taken in conjunction with the accompanying drawings.